CN103493768A - Method for obtaining holonephros expression green fluorescent protein white zebra fish - Google Patents
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Abstract
The invention discloses a method for obtaining a holonephros expression green fluorescent protein zebra fish. The method can overcome the defects that green fluorescent protein of an existing zebra fish line can not be evenly and simultaneously expressed in a whole original glomerulus, a tubule and a catheter. Green fluorescent protein of a zebra fish in the method can be simultaneously expressed in a whole original glomerulus, an original kidney tubule and an original kidney catheter. The method is a crossbreeding method according to which an Na, K-ATPase alpha1A4: GFP line zebra fish and a wt1b: GFP line zebra fish are mutual parents. The method for obtaining the holonephros expression green fluorescent protein zebra fish includes the following steps of hybridization parent selection and breeding, matching and breeding, hybridization character sieving, hybridization filial generation breeding and hybridization character fixing and retaining. The method for obtaining the holonephros expression green fluorescent protein zebra fish is a separation element hybridization stable integration method, the implementation process is easy to control, a zebra fish line of which green fluorescent protein can be simultaneously, evenly and efficiently expressed in a whole original glomerulus, an original kidney tubule and an original kidney catheter is obtained and the requirement for integrality and completeness of study on the original kidney functions of the zebra fish is met.
Description
Technical field
The present invention relates to a kind of zebra fish breeding method, particularly relate to a kind of breeding method that obtains holonephros expressing green fluorescent protein zebra fish, belong to biological technical field.
Background technology
1962, Shimomura etc. have found that bioluminescent protein matter is aequorin (aequorin) first in Victoria's multitube jellyfish (Aequoria victoria), and observe a kind of accessory substance-green fluorescent protein (Green Fluorescent Protein, GFP) issue light green color fluorescence that irradiates in ultraviolet ray.1974, Shimomura etc. completed the GFP purifying, and after 18 years, Prasher etc. clones its gene, and Chalfie in 1994 etc. have realized the expression of GFP in Bacillus coli cells and Caenorhabditis elegans afterwards.GFP is as a kind of important biomarker albumen, can autocatalysis form chromonic structures and ultraviolet or blue-light excited under send green fluorescence.Because GFP possesses that fluorescence is stable, the safety non-toxic evil, be easy to detect and be easy to obtain numerous advantages such as mutant, thereby be widely used in the research fields such as cell biology, molecular biology and microorganism.
Growth and the mankind of zebra fish archinephridium are basically identical, and it is extremely similar that the groups of cells of its nephron or glomerulus becomes, and have advantage simple in structure, so the researcher often studies human kidney's growth and function using zebra fish as desirable model organism.The archinephridium of zebra fish is comprised of 2 nephrons, and the pronephric duct that 2 renal tubules will be positioned at respectively both sides is connected with the glomerulus merged, and pronephric duct converges and is communicated to cloaca at afterbody.After fertilization 2～3 days, had the archinephridium structure that can complete the osmotic adjustment function in zebrafish embryo.In order to study better zebra fish kidney growth course, the zebra fish strain that development GFP kidney specific is expressed is significant.2007, the people such as Yan Liu produce Na, K-ATPase alpha1A4:GFP transgenic strain zebra fish, realized specific expressed in zebra fish pronephric tubule and former nephridioduct of GFP, but have special green fluorescence in the former glomerulus of this strain zebra fish, expresses.In the same year, Perner etc. utilize transgenic technology to develop wt1b:GFP strain zebra fish.At this transgenic zebrafish strain after fertilization 35h, can observe in the former nephridioduct of its former glomerulus and proximal part and send green fluorescence, but can not observe special GFP at distal catheter, express.When above-mentioned two kinds of strain zebra fishs are studied for archinephridium, GFP can not express in whole former glomerulus, tubule and conduit simultaneously equably, thereby has affected globality and the integrality of the former renal function study of zebra fish.
Summary of the invention
Purpose of the present invention is exactly for the deficiencies in the prior art, and a kind of method that obtains the zebra fish that green fluorescent protein expresses in former glomerulus, pronephric tubule and former nephridioduct simultaneously is provided.
For achieving the above object, technical scheme of the present invention is as follows:
A kind of method that obtains holonephros expressing green fluorescent protein zebra fish is characterized in that: be with Na, K-ATPase alpha1A4:GFP strain zebra fish and wt1b:GFP strain zebra fish be parent's cross breeding method each other.
Na, K-ATPase alpha1A4:GFP strain zebra fish is to present the specific expressed zebra fish strain of green fluorescent protein in a kind of pronephric tubules that obtain via artificial culture and pronephric duct, and wt1b:GFP strain zebra fish is that the zebra fish strain of part egfp expression is arranged in a kind of glomeruli of pronephros obtained via artificial culture, pronephric tubules.The technical program is to utilize Na, K-ATPase alpha1A4 promotor and the complementation of wt1b promotor tissue specific expression, by the resolution element hybrid stability, integrate, different egfp expression proterties centralized integration in addition by it, thus obtain the zebra fish strain with new properties and characteristics.The zebra fish product that obtain tie up in archinephridium bead, pronephric tubule and former nephridioduct possesses the egfp expression proterties simultaneously.
According to conventional crossbreeding method of operating, the inventive method according to the breeding of hybrid strain seed selection, combo, hybridization character screening, hybrid generation cultivate, the hybridization proterties is fixing with retain several steps is implemented.In parent's seed selection, select the parent zebra fish that reaches sexual maturity and there is good egfp expression proterties, artificial feeding is as the female milter of parent in addition.In the combo breeding, adopt conventional combo propagation method to obtain zebra fish fertilized egg.In the hybridization character screening, in the expression of fluorescence microscopy Microscopic observation fertilized egg green fluorescence protein gene, pick out the zebrafish embryo that fluorescence display is all arranged in glomeruli of pronephros, tubule and conduit, take further hybrid generation to cultivate.In hybrid generation is cultivated, adopt rearing method to obtain hybridization first filial generation F1.In the hybridization proterties is fixed and is retained, obtain the holonephros expressing green fluorescent protein zebra fish of genetic stability by the hybrid generation selfing, again by itself and common zebra fish test cross, sift out parent zebra fish corresponding to 100% holonephros expressing green fluorescent protein embryo as the preservation of reserving seed for planting of two integration pure lines.
Technique scheme can be taked the artificial propagation means of optimizing, and the steps such as hybrid strain seed selection, combo breeding, hybrid generation cultivation is optimized, with acquisition green fluorescent protein uniform high-efficiency ground specific expressed zebra fish strain in holonephros.Specifically comprise:
The hybrid strain seed selection: zebra fish is raised in circulating water culture system, and the photoperiod is daytime 14h, dark 10h, 26 ℃～29 ℃ of water temperatures; Throw something and feed every day live body fairy shrimp or prawn slice 3 times, cleaning and fish excrement, foreign material and overfeeding after feeding 5min.Raise after certain hour as the female milter of parent.
Combo breeding: obtaining zebra fish fertilized egg method is: the female milter of the parent of quantity 2:1 is put into mating box isolate forster, and the mating box is placed in 26 ℃～29 ℃ isoperibols and carries out dark incubated overnight, and the photoperiod is daytime 14h, dark 10h; Cultivate to finish, mix the male and female parent population, the fenced in internal layer mating in bottom box slant setting on outer mating box and be placed in 26 ℃～29 ℃ waters bath with thermostatic control, is treated to Gunther parent fish spawning; After Gunther parent fish spawning 25min～35min, take out parent population, select the fertilized egg of collecting mating box bottom.
Hybridization character screening: detect the expression effect of green fluorescent protein spot gene after fertilized egg is cultivated 24h～28h in 26 ℃～29 ℃ constant temperature water aerations, select the zebrafish embryo that fluorescence is all arranged in glomeruli of pronephros, tubule and conduit and carry out the hybrid generation cultivation.
Hybrid generation is cultivated: after fertilization the 1d～4d: described zebrafish embryo is cultivated at 26 ℃～29 ℃ environment constant temperature, obtains juvenile fish; After fertilization the 5d～9d: juvenile fish feeding yolk water, every day, feeding was 2 times; After fertilization the 10d～16d: juvenile fish mixes the yolk water of feeding fairy shrimp and filter screen grinding, and concrete grammar is first to juvenile fish, to raise in container and drop into a small amount of fairy shrimp, the yolk water that adds again filter screen to grind after 25min～35min, and every day, feeding was at least 2 times; After after fertilization 17d: juvenile fish feeding fairy shrimp, every day, feeding was 2 times, changed half water of breeding fish every day.Be cultured to after fertilization and within 3 months, obtain hybridization first filial generation F1.
The hybridization proterties fixing with retain: hybridization first filial generation F1 goes down to posterity and cultivates the holonephros expressing green fluorescent protein zebra fish that obtains genetic stability through selfing, and selfing is gone down to posterity while cultivating to adopt and cultivated identical cultivation raising method with hybrid generation and obtain certainly for filial generation; Selfing F3 zebra fish and common zebra fish test cross, filter out parent zebra fish corresponding to 100% holonephros expressing green fluorescent protein embryo and be sheerly as two integration the preservation of reserving seed for planting.
The present invention is a kind of method that resolution element hybrid stability is integrated, and its technique effect and traditional hybrid vigour are distinguished to some extent.After the conventional hybridization advantage often refers to that plant that two kinds of hereditary basiss are different or animal are hybridized, the various proterties that its filial generation shows all are better than hybridizing parents.The inventive method is that the promotor tissue specific expression is in addition complementary, by the resolution element hybrid stability, integrates, and obtains the filial generation that tissue specificity is expressed simultaneously.This method goes for concentrating and integrating of other multiple transgenic zebrafish transgenosis proterties equally.
Compared with prior art, the invention has the beneficial effects as follows: (1) method implementation process is simple and easy to control; (2) method can obtain green fluorescent protein in former glomerulus, pronephric tubule and former nephridioduct simultaneously and the zebra fish strain expressed of uniform high-efficiency; (3) gained zebra fish strain is for the research of the former renal function of zebra fish, utilize the expression of green fluorescent protein in the live body fish can observe at any time the archinephridium egfp expression feature of when phase in office, can meet globality and integrality requirement to the former renal function study of zebra fish.
The accompanying drawing explanation
Fig. 1 is parent Na, and K-ATPase alpha1A4:GFP strain zebra fish is at 48 hours fluorescence pictures of after fertilization.
Fig. 2 is that parent wt1b:GFP strain zebra fish is at 48 hours fluorescence pictures of after fertilization.
Fig. 3 is that the hybrid generation zebra fish that obtains of the present invention is at 48 hours fluorescence pictures of after fertilization.
Fig. 4 is that the hybrid generation zebra fish that obtains of the present invention is in 48 hours white light field picture of after fertilization.
Fig. 5 is to the development impact fluorescence picture of zebra fish archinephridium after Apex1Morpholino (MO) injection.(upper zebra fish is contrast MO injection, and lower zebra fish is the Apex1MO injection)
Fig. 6 is to the development impact white light field picture of zebra fish archinephridium after Apex1Morpholino (MO) injection.(upper zebra fish is contrast MO injection, and lower zebra fish is the Apex1MO injection)
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are further described.
Embodiment mono-
As shown in Figure 1 to 4, adopt the inventive method to obtain holonephros expressing green fluorescent protein zebra fish.
1, hybrid strain seed selection
Select Na, K-ATPase alpha1A4:GFP strain zebra fish and wt1b:GFP strain zebra fish are used parent population as hybridization.Zebra fish as the parent has passed through fluorescent screening in embryonic period, embryonic phase or young stage, guarantee Na, present the specific expressed of green fluorescent protein in the renal tubule of K-ATPase alpha1A4:GFP strain parent population and pronephric duct, the expression of part green fluorescent protein is arranged in wt1b:GFP strain parent population glomeruli of pronephros, pronephric tubules.Fig. 1 is parent Na, and K-ATPase alpha1A4:GFP strain zebra fish is at 48 hours fluorescence pictures of after fertilization; Fig. 2 is that parent wt1b:GFP strain zebra fish is at 48 hours fluorescence pictures of after fertilization.
Select Na, male, female each 10 of K-ATPase alpha1A4:GFP strain zebra fish, wt1b:GFP strain zebra fish female, male each 10 as parent population.Parent population has all reached sexual maturity, and should possess that the adult fish bodily form is normal, body colour is shinny, fish scale and fin does not have breakage, the predation flexibly of moving about is quick, there is no the desirable features such as abnormal behaviour.
40 parent populations that choose are raised in circulating water culture system, are guaranteed that the photoperiod is: daytime 14h, dark 10h; 26 ℃～29 ℃ of water temperatures; Throw something and feed every day live body fairy shrimp or prawn slice 3 times, the foreign material such as the food of not eating up after feeding 5min～10min and fish excrement are cleared up sucking-off in time.
2, combo breeding
Dividing two groups is bred: in first group, and Na, K-ATPase alpha1A4:GFP zebra fish is as male parent, and wt1b:GFP strain zebra fish is as female parent; In second group, Na, K-ATPase alpha1A4:GFP zebra fish is as female parent, and wt1b:GFP strain zebra fish is as male parent.
Laying eggs eve, and in two groups, selecting respectively 2 rauns and 1 milter (female milter ratio 2:1) to put into the mating box, inserting transparent baffle, and making female milter lay respectively at the baffle plate both sides.Afterwards the mating box is placed in to 26 ℃～29 ℃ isoperibols and carries out the dark raising of spending the night, the photoperiod is 14h in daytime, dark 10h.
Cultivate to finish, take out transparent baffle morning next day, the fenced in internal layer mating in bottom box slant setting on outer mating box and be placed in 26 ℃～29 ℃ waters bath with thermostatic control, is kept to the surrounding environment peace and quiet, allow female milter chase and to lay eggs voluntarily.Fish-egg is fallen into outer mating box bottom through the fence of internal layer mating box.
The process of chasing is carried out about 30min, and parent population is taken out, and selects the fertilized egg of collecting mating box bottom, and is placed in the culture dish that is placed with clean water aeration and is cultivated in 26 ℃～29 ℃ constant incubators; Early, respectively change water once, and the embryo who dies is removed in time evening.
3, hybridization character screening
Fertilized egg is used fluorescence microscope to detect the expression effect of green fluorescent protein spot gene cultivate 24h in 26 ℃～29 ℃ constant temperature water aerations after; Select the zebrafish embryo that fluorescence is all arranged in glomeruli of pronephros, tubule and conduit and carry out the hybrid generation cultivation.The embryo who will there is no fluorescence or die removes from culture dish.
4, hybrid generation is cultivated
The desirable embryo that the hybridization character screening is obtained normally cultivates, and concrete steps are as follows:
After fertilization the 1d～4d:
Clean water aeration is rejected and changed to the embryo or the juvenile fish that die, and the temperature of fresh water should be consistent with former water, guarantees that zebrafish embryo is cultivated until obtain juvenile fish in the stable environment of 26 ℃～29 ℃ of constant water temperatures.
After fertilization the 5d～9d:
This, juvenile fish can move about and opening in water in period, can start feeding yolk water.Yolk water is by the egg the boiled taking-up yolk of peeling off, and after grinding, meticulous mesh filter screen (0.2mm aperture) adds water and makes.Draw and splash in right amount in juvenile fish raising container with glue head dropper, every day, feeding was 2 times.After feeding yolk water, the water quality of juvenile fish existence easily changes, so at least changes water once every day, and the unnecessary yolk of cleaning container bottom.
After fertilization the 10d～16d:
Start to mix throw something and feed fairy shrimp and yolk water from after fertilization 10d.During feeding, first feed a small amount of fairy shrimp, wait the 30min left and right and feed again a small amount of yolk water, guarantee that all juvenile fish all can take food.Every day, feeding was at least 2 times.Change water every day 1～3 time, change half at every turn and raise water.
After after fertilization 17d
From after fertilization 17d, can a feeding fairy shrimp, every day 2 times.Change half every day and raise water.
The juvenile fish fairy shrimp of can successfully taking food, and health is grown up rapidly, resistance strengthens, and it can be moved into to circulating water culture system, together with adult fish, raises.
5, the hybridization proterties is fixed and is retained
Hybrid generation was cultivated to after fertilization after 3 monthly ages, adopted selfing to go down to posterity and cultivated fixing hybridization proterties, obtained the hybrid generation of genetic stability.The employing selfing adopts the method consistent with aforementioned hybrid generation breeding method to obtain filial generation in going down to posterity and cultivating.Fig. 3 is that the hybrid generation zebra fish that obtains of the present invention is at 48 hours fluorescence pictures of after fertilization; Fig. 4 is that the hybrid generation zebra fish that obtains of the present invention is in 48 hours white light field picture of after fertilization.
3 generations of selfing and after, by the holonephros expressing green fluorescent protein zebra fish of genetic stability and common zebra fish test cross, filter out parent zebra fish corresponding to 100% holonephros expressing green fluorescent protein embryo and be sheerly as two integration the preservation of reserving seed for planting.
Embodiment bis-
Adopt the inventive method to obtain holonephros expressing green fluorescent protein zebra fish.Itself and embodiment mono-something in common no longer repeat, and its difference is:
3, hybridization character screening
Fertilized egg is used fluorescence microscope to detect the expression effect of green fluorescent protein spot gene cultivate 28h in 26 ℃～29 ℃ constant temperature water aerations after; Select the zebrafish embryo that fluorescence is all arranged in glomeruli of pronephros, tubule and conduit and carry out the hybrid generation cultivation.The embryo who will there is no fluorescence or die removes from culture dish.
Embodiment tri-
Adopt the inventive method to obtain holonephros expressing green fluorescent protein zebra fish.Itself and embodiment mono-something in common no longer repeat, and its difference is: each stage cultivates 26 ℃～29 ℃ of water temperature conditions and is adjusted into 28.5 ℃.
Embodiment tetra-
Adopt the inventive method to obtain holonephros expressing green fluorescent protein zebra fish.Its part identical with embodiment bis-no longer repeats, and its difference is: each stage cultivates 26 ℃～29 ℃ of water temperature conditions and is adjusted into 28.5 ℃.
Test example one
As shown in Figure 5, Figure 6, the holonephros expressing green fluorescent protein zebrafish embryo that the inventive method obtains is applied to the test of kidney gene functional research.
The holonephros expressing green fluorescent protein zebrafish embryo (after fertilization 3d) that utilizes that utilizes embodiment tetra-breedings to obtain is material, carries out and affects the related gene functional study that kidney is grown.Fig. 5, Fig. 6 grow teratogenic picture to the zebra fish archinephridium after Apex1Morpholino (MO) injection.Fig. 5, Fig. 6 show that the growth of control group zebra fish archinephridium is normal, and Apex1 strikes low group kidney duct and shortens and deformity, and this growth that shows Apex1 gene pairs kidney has important function.
Claims (10)
1. a method that obtains holonephros expressing green fluorescent protein zebra fish, it is characterized in that: be with Na, K-ATPase alpha1A4:GFP strain zebra fish and wt1b:GFP strain zebra fish be parent's cross breeding method each other.
2. method according to claim 1, it is characterized in that: detect the expression effect of green fluorescence protein gene after fertilized egg is cultivated 24h～28h in 26 ℃～29 ℃ constant temperature water aerations, select the zebrafish embryo that fluorescence is all arranged in glomeruli of pronephros, tubule and conduit and carry out the hybrid generation cultivation.
3. method according to claim 3 is characterized in that: fertilized egg is cultivated after 24h the expression effect that detects green fluorescence protein gene in 26 ℃～29 ℃ constant temperature water aerations.
4. it is characterized in that according to the method in claim 2 or 3: described hybrid generation breeding method is:
After fertilization the 1d～4d: described zebrafish embryo is cultivated at 26 ℃～29 ℃ environment constant temperature, obtains juvenile fish,
After fertilization the 5d～9d: juvenile fish feeding yolk water, every day, feeding was 2 times,
After fertilization the 10d～16d: juvenile fish mixes feeding fairy shrimp and yolk water, and concrete grammar is first to juvenile fish, to raise in container and drop into a small amount of fairy shrimp, the yolk water that adds again filter screen to grind after 25min～35min, and every day, feeding was at least 2 times,
After after fertilization 17d: juvenile fish feeding fairy shrimp, every day, feeding was 2 times, changed half every day and raised water.
5. method according to claim 4, it is characterized in that: described cultivation water temperature condition is 28 ℃.
6. method according to claim 5 is characterized in that: hybrid generation is cultivated the hybrid generation zebra fish obtained and is gone down to posterity and cultivate the holonephros expressing green fluorescent protein zebra fish that obtains genetic stability through selfing.
7. method according to claim 6, it is characterized in that: the holonephros expressing green fluorescent protein zebra fish of described genetic stability and common zebra fish test cross filter out parent zebra fish corresponding to 100% holonephros expressing green fluorescent protein embryo and are sheerly as two integration the preservation of reserving seed for planting.
8. method according to claim 1, it is characterized in that: the fertilized egg acquisition methods is:
The female milter of the parent of quantity 2:1 is put into mating box isolate forster, and the mating box is placed in 26 ℃～29 ℃ isoperibols and carries out dark incubated overnight, and the photoperiod is daytime 14h, dark 10h,
Cultivate to finish mix the male and female parent population, the fenced in internal layer mating in bottom box slant setting on outer mating box and be placed in 26 ℃～29 ℃ waters bath with thermostatic control, treated to Gunther parent fish spawning,
After Gunther parent fish spawning 25min～35min, take out parent population, select the fertilized egg of collecting mating box bottom.
9. method according to claim 1, it is characterized in that: the zebra fish as the parent is raised in circulating water culture system, and the photoperiod is daytime 14h, dark 10h, 26 ℃～29 ℃ of water temperatures; Throw something and feed every day live body fairy shrimp or prawn slice 3 times, cleaning fish excrement, foreign material and overfeeding after feeding 5min.
10. the described method of claim 8 or 9, it is characterized in that: described cultivation water temperature condition is 28 ℃.
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